Formulation of modified interleukin-7 fusion protein

ABSTRACT

Provided is a pharmaceutical formulation comprising a modified IL-7 protein. More particularly, it comprises (a) a modified IL-7 fusion protein; (b) a basal buffer with a concentration of 10 to 50 mM; (c) a sugar with a concentration of 2.5 to 5 w/v %; and (d) a surfactant with a concentration of 0.05 to 6 w/v %. Such pharmaceutical formulation of a modified IL-7 fusion protein does not show aggregates formation, but shows protective effects on proteins under stress conditions such as oxidation or agitation, and thus can effectively be used for the treatment of a patient.

TECHNICAL FIELD

The present invention relates to a formulation of a pharmaceuticalcomposition comprising a modified interleukin-7 fusion protein.

BACKGROUND ART

Interleukin-7 (IL-7) is a cytokine stimulating immune responses mediatedby T cell and B cell, and specifically, it plays an important role in anadaptive immune system. Although IL-7 is mainly secreted by stromalcells in the bone marrow and the thymus, it is also produced inkeratinocytes, dendritic cells, hepatocytes, neural and epithelial cells(Heufler C et al., 1993, J. Exp. Med. 178 (3): 1109-14; Kroncke R etal., 1996, Eur. J. Immunol. 26 (10): 2541-4: Sawa Y et al., 2009,Immunity 30 (3): 447-57; Watanabe M et al., 1995, J. Clin. Invest. 95(6): 2945-53).

IL-7 actives the immune system by influencing the survival anddifferentiation of T cell and B cell, and stimulating the activity of NK(natural killer) cell, and so on, and specifically, it plays animportant role in the development of B cell. IL-7 enhances the immuneresponse in the human body by stimulating the secretion of IL-2, a typeof cytokine, and interferon-γ.

In other words, IL-7 is a cytokine which promotes the survival andproliferation of T-cell, B-cell and other immune cells, and is anexcellent candidate for an immune therapeutic agent applicable tovarious disorders such as viral infection, cancer and immune systemdamage. Recently a clinical study has been conducted to elucidate theeffect by IL-7 on various malignancies and human immune deficiency virusinfection, and as a result, an immune enhancing effect in human of IL-7was reported (Fry T J et al., 2002, Blood 99 (11): 3892-904; Muegge K etal., 1993, Science 261 (5117): 93-5; Rosenberg S A et al., J.Immunother. 29 (3): 313-9). In addition, IL-7 has been reported to behelpful in the immune recovery after allogenic stem cell transplantation(Snyder K M, 2006, Leuk Lymphoma 47 (7). 1222-8), and is used for thetreatment of lymphopenia as well. Accordingly, IL-7 reorganizes immunecells, enhances T cell function, and competes with the substances whichinduce immunosuppression, and thus it can be used for treating cancer orchronic infection.

However, in order to utilize proteins such as IL-7 for a pharmaceuticaluse, it is necessary to prepare a suitable formulation in considerationof the structure and the physiochemical stability in surroundingenvironment of the protein. In addition, the physical and chemicalstability of the protein is affected by external factors such asmanufacturing of a pharmaceutical formulation, purification of aprotein, and storage. In general, a protein is structurally andthermodynamically unstable, and thus is easily subject to aggregatesformation or physiochemical degradation. In addition, when Fc region ofan immunoglobulin is used as a fusion partner, the physiochemicalproperty of the entire fusion protein such as thermodynamic property,Zeta potential and stress stability, etc., can vary greatly since the Fcregion has a large molecular weight and a complex structure.Specifically, little is known with respect to a stabilized formulationfor the modified IL-7 fusion protein. Thus, it is necessary to optimizethe formulation of a protein in order to increase the stability of theprotein for a pharmaceutical use.

DISCLOSURE OF INVENTION Technical Problem

Therefore, the present inventors have established an appropriateformulation to increase the stability of a modified IL-7 fusion proteinfor use in treating a variety of disorders.

An object of the present invention is to provide a pharmaceuticalformulation comprising a modified IL-7 fusion protein with increasedstability.

Solution to Problem

In accordance with the object of the present invention, there isprovided a pharmaceutical formulation comprising: (a) a modified IL-7fusion protein; (b) a basal buffer with a concentration of 10 to 50 mM;(c) a sugar with a concentration of 2.5 to 5 w/v %; and (d) a surfactantwith a concentration of 0.05 to 6 w/v %.

Advantageous Effects of Invention

A pharmaceutical formulation of a modified IL-7 fusion protein accordingto the present invention does not show aggregates formation, andmaintains stability of the modified IL-7 fusion protein under stressconditions such as oxidation or agitation, and thus can be used for thetreatment of a patient.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the structure of a modified IL-7fusion protein according to the present invention.

FIG. 2 presents graphs showing the results of differential scanningcalorimetry (DSC) of a modified IL-7 fusion protein under various basalbuffer conditions.

FIG. 3 presents graphs showing the results of dynamic light scattering(DLS) analysis of a modified IL-7 fusion protein under various basalbuffer conditions.

FIG. 4 presents graphs showing the results of DLS analysis of a modifiedIL-7 fusion protein under the conditions in which various excipientswere added to a histidine-acetate buffer.

FIG. 5 presents graphs showing the results of DLS analysis of a modifiedIL-7 fusion protein under the conditions in which various excipientswere added to a sodium citrate buffer.

MODE FOR THE PRESENT INVENTION

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical formulation comprising(a) a modified IL-7 fusion protein; (b) a basal buffer with aconcentration of 10 to 50 mM; (c) a sugar with a concentration of 2.5 to5 w/v %; and (d) a surfactant with a concentration of 0.05 to 6 w/v %.

As used herein, the term “interleukin-7 (IL-7)” refers to ahematopoietic growth factor, which is expressed in or secreted byvarious stromal cells, keratin-producing cells, dendritic cells,neurons, epidermal cells, etc. IL-7 may promote an immune response bybinding to a receptor, and activate a variety of immune cells such as Tcell, B cell, mononuclear cell involved in the immune system.

Such IL-7 is either interleukin-7 (IL-7) or a polypeptide having asimilar activity to IL-7.

IL-7 the present invention includes the polypeptides consisting of theamino acid sequences represented by SEQ ID NOs:1 to 6. In addition, theamino acid sequence of the IL-7 fusion protein may have a sequencehomology of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore to the sequences of SEQ ID NOs:1 to 6. IL-7 may be an IL-7 fusionprotein or a fusion protein comprising a fragment thereof. In addition,IL-7 may be derived from human, rat, mouse, monkey, cow or sheep.

Specifically, human-derived IL-7 may have the amino acid sequencerepresented by SEQ ID NO:1 (Genbank Accession No. P13232); rat-derivedIL-7 may have the amino acid sequence represented by SEQ ID NO:2(Genbank Accession No. P56478); mouse-derived IL-7 may have the aminoacid sequence represented by SEQ ID NO:3 (Genbank Accession No. P10168);monkey-derived IL-7 may have the amino acid sequence represented by SEQID NO:4 (Genbank Accession No. NP_001279008); bovine IL-7 may have theamino acid sequence represented by SEQ ID NO:5 (Genbank Accession No.P26895); and sheep-derived IL-7 may have the amino acid sequencerepresented by SEQ ID NO:6 (Genbank Accession No. Q28540).

As used herein, the term “modified IL-7” refers to a molecule wherein anoligopeptide composed of 1 to 10 amino acids is present at the terminalof IL-7, thereby to have a different sequence from the wild type IL-7.

Preferably, a modified IL-7 according to the present invention has thefollowing structure:

A-IL-7;

wherein said A may be bound to the N-terminal of the IL-7. The nucleicacid encoding said A may be present with IL-7 genes sequentially on aDNA construct, which may be expressed as a protein. Further, said A maybe expressed or synthesized independently, and then bound to IL-7 by achemical bond.

Said A may comprise 1 to 10, specifically 2 to 8, more specifically 1 to5 amino acids, wherein the comprised amino acid may be selected from thegroup consisting of methionine (M), glycine (G), and a combinationthereof. Examples of said A include M, G, MM, MG, GM, GG, MMM, GMM, MGM,MMG, GGM, GMG, MGG, GGG, MMMM (SEQ ID NO:9), GMMM (SEQ ID NO:10), MGMM(SEQ ID NO:11), MMGM (SEQ ID NO:12), MMMG (SEQ ID NO:13), GGMM (SEQ IDNO:14), MGGM (SEQ ID NO:15), MMGG (SEQ ID NO:16), GMGM (SEQ ID NO:17),MGMG (SEQ ID NO:18), GMMG (SEQ ID NO:19), GGGM (SEQ ID NO:20), MGGG (SEQID NO:21), GMGG (SEQ ID NO:22), GGMG (SEQ ID NO:23), GGGG (SEQ IDNO:24), MMMMM (SEQ ID NO:25), GMMMM (SEQ ID NO:26), GGMMM (SEQ IDNO:27), GGGMM (SEQ ID NO:28), GGGGM (SEQ ID NO:29), MGMMM (SEQ IDNO:30), MGGMM (SEQ ID NO:31), MGGGM (SEQ ID NO:32), MGGGG (SEQ IDNO:33), MMGMM (SEQ ID NO:34), MMGGM (SEQ ID NO:35), MMGGG (SEQ IDNO:36), MMMGM (SEQ ID NO:37), MMMGG (SEQ ID NO:38), MMMMG (SEQ IDNO:39), MGGGM (SEQ ID NO:40), MGMGM (SEQ ID NO:41), GMGMG (SEQ IDNO:42), GMMMG (SEQ ID NO:43), GGMGM (SEQ ID NO:44), GGMMG (SEQ IDNO:45), MGGMG (SEQ ID NO:46), MGMGG (SEQ ID NO:47), GMMGM (SEQ IDNO:48), MGMMG (SEQ ID NO:49), GMGGM (SEQ ID NO:50), MMGMG (SEQ IDNO:51), GMMGG (SEQ ID NO:52), GMGGG (SEQ ID NO:53), GGMGG (SEQ IDNO:54), GGGMG (SEQ ID NO:55) and GGGGG (SEQ ID NO:56), etc. According toan example of the present invention, said A may be M, MM, GM, MGM orMMM.

A modified IL-7 fusion protein according to the present invention mayfurther comprise the Fc region of a modified immunoglobulin at theC-terminal thereof.

As used herein, the term “modified IL-7 fusion protein” refers to amolecule wherein a modified immunoglobulin Fc region is bound to theC-terminal of a modified IL-7.

Said modified immunoglobulin Fc region may be one whose binding activityto a Fc receptor or to a complement is modified, resulting in weakenedantibody-dependent cellular cytotoxicity (ADCC) or complement dependentcytotoxicity (CDC). Such modification may be achieved by geneticmutation (i.e., substitution, deletion of the sequence) of the bindingsite for Fc gamma receptor (FcrR), C1q, and Fc binding receptor (FcRn)in the Fc region.

In addition, such modification may be achieved by mixing different typesof immunoglobulin sequences, that is, by preparing a hybrid Fc.

As used herein, the term “Fc region”, “Fc fragment” or “Fc” may includea hinge region, a heavy chain constant region 2 (CH2) domain, and aheavy chain constant region 3 (CH3) domain in the direction from theN-terminal to the C-terminal. Such region may further comprise a hingeregion, in which case the hinge region may serve as a linker, and mayappropriately be modified to improve the property of the molecule.

The Fc region or a fragment thereof referred to as “hFc” or “hyFc” is atype of hybrid Fc, which is prepared by combining different types of Fcregions. Such hyFc may include a portion of a human IgD hinge region, anamino acid residue of a human IgD CH2 domain, an amino acid residue of ahuman IgG4 CH2 domain, and an amino acid residue of a human IgG4 CH3domain.

The Fc region variant may be modified so as to prevent truncation at thehinge region. Specifically, the amino acid at position 144 and/or theamino acid at position 145 of SEQ ID NO: 7 can be modified. Preferably,K, the amino acid at position 144 of SEQ ID NO:7, may be substitutedwith G or S, and E, the amino acid at position 145 of SEQ ID NO:7, maybe substituted with G or S, to form a variant.

In addition, two fusion proteins may form one dimer. For example, whenthe third domain is Fc region, the Fc regions may bind to each other toform a dimer.

According to an example of the present invention, the modifiedimmunoglobulin Fc region may be a polypeptide consisting of the aminoacid sequence represented by SEQ ID NO:7. The Fc region of a modifiedimmunoglobulin according to the present invention may be one describedin U.S. Pat. No. 7,867,491, and can be producted with reference to thedescription in U.S. Pat. No. 7,867,491.

According to an example of the present invention, the modified IL-7 mayhave the amino acid sequence of SEQ ID NO:8. In addition, the modifiedIL-7 may have a sequence homology of 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% to the amino acid sequence of SEQ ID NO:8,

As used herein, the term “basal buffer” refers to a solution whichendures a pH change by the action of an acid-base conjugate componentcontained therein.

A basal buffer according to the present invention may behistidine-acetate or sodium citrate solution. The basal buffer may beused in a concentration of 10 to 50 mM, 15 to 40 mM, or 20 to 30 mM.According to an example of the present invention, it may be used in aconcentration of 20 mM.

The histidine-acetate solution is a buffer containing histidine ions.The histidine-acetate solution is prepared by titrating L-histidine withliquid acetic acid. Meanwhile, the sodium citrate solution is preparedby titrating the sodium citrate solution with hydrous citric acidregarding the final concentration and pH of the solution.

A sugar stabilizes the fusion protein in the present invention. Inaddition, a sugar is widely used as an excipient for pharmaceuticalformulations as it is easily dissolved in water and has a sweet taste.

In a formulation according to the present invention, the sugar may becomprised in a concentration of 2.5 to 5 w/v %, 3.5 to 5 w/v %, or 4.5to 5 w/v %, and particularly 5 w/v % in an example of the presentinvention.

In a formulation according to the present invention, the sugar may besucrose, trehalose, dextrose, or a mixture thereof. In one example ofthe present invention, the sugar may be sucrose. Sucrose is adisaccharide formed of glucose and fructose by 1, 2-binding, which isrelatively stable in an alkaline environment but may be easilyhydrolyzed by an acid.

In a pharmaceutical formulation according to the present invention, asugar has the effect of protecting a fusion protein from the oxidationstress, and a sugar alcohol may be used as a sugar substitute or anauxiliary substance. The sugar alcohol may be sorbitol, xylitol,maltitol, mannitol or a mixture thereof. In one example of the presentinvention, the sugar alcohol may be sorbitol.

As used herein, the term “surfactant” is a surface activating agent, andspecifically, it refers to a non-ionic surfactant. In the pharmaceuticalformulation according to the present invention, the surfactant inhibitsaggregates formation of a fusion protein, thereby increasing thestability of the fusion protein.

In the formulation according to the present invention, a surfactant maybe added in a concentration of 0.05 to 6 w/v %, 0.1 to 5.5 w/v %, or 0.1to 5.0 w/v %.

The surfactant may be polysorbate, polyoxyethylene alkyl ether,polyoxyethylene stearate, alkyl sulfate, polyvinyl pyridone, poloxameror a mixture thereof. Specifically, the surfactant may be polysorbate orpoloxamer.

The polysorbate according to the present invention is a polyoxyethylenehigher aliphatic alcohol formed by combinding sorbitan fatty acid esterwith ethylene oxide, which may be polysorbate 20 (monolaurate), 40(monopalmitate), 60 (monostearate), 65 (tristearate), or 80(mono-oleate) based on the number of the polyoxyethylene group and thekind of the fatty acid. In addition, poloxamer is apolyoxypropylene-polyoxyethylene block copolymer, which may varydepending on the length of the polymer.

In one example of the present invention, the surfactant may bepolysorbate 20, polysorbate 80 or poloxamer 188.

A pharmaceutical formulation according to the present invention mayfurther comprise an amino acid such as arginine, glutamate, glycine,histidine or a mixture thereof, etc. The amino acid is comprised as anexcipient in order to stabilize a protein, and various amino acids maybe comprised depending on the type of the protein. The amino acid may bearginine, glutamate, glycine or histidine, and according to an exampleof the present invention, it may be glutamate. The amino acid may beadded in a concentration of 40 to 60 mM, according to an example of thepresent invention, 50 mM.

When using a pharmaceutical formulation of the present invention as aninjection, it may further comprise a sugar alcohol to adjust the osmoticpressure. The sugar alcohol refers to a long-chain alcohol in which acarbonyl group of a sugar is reduced to a hydroxyl group (—OH). Thesugar alcohol may be added such that the concentration thereof reaches 1to 2 w/v %, and according to an example of the present invention, 1.5w/v %.

Examples of sugar alcohol for use in the present invention includesorbitol, xylitol, maltitol, mannitol or a mixture thereof. According toan example of the present invention, the sugar alcohol may be sorbitol.Sorbitol is a sugar alcohol having 6 hydroxyl groups formed by highpressure addition and reduction of glucose, which is also referred to asD-sorbitol or D-glucitol.

The pharmaceutical formulation of the present invention may have a pH of5.0 to 6.0, specifically 5.0. The conformational stability of a fusionprotein decreases if the pH of the basal buffer is lower than 5.0, whilethe fusion protein may show aggregates formation depending on a specificcomposition if the pH exceeds 6.0.

The formulation may be a liquid formulation.

The inventors have prepared a modified IL-7 fusion protein in which anoligopeptide is bound to the N-terminal of IL-7 and the Fc region of animmunoglobulin is bound to the C-terminal of IL-7, and selected a basalbuffer and an excipient to prepare a pharmaceutical formulation of thefusion protein

First, stability of the modified IL-7 fusion protein depending on abasal buffer was examined at various pH conditions. When sodium citratewas used as a basal buffer, stability of the fusion protein increased asthe pH increased (Table 2 and FIG. 2), but the zeta potential decreasedand aggregates of the fusion protein were formed as the pH increased.When histidine-acetate was used as a basal buffer, similar results wereobtained, and thus, the experiment was conducted at the condition of pH5.0 first (Table 3 and FIG. 3).

In addition, to examine the protective effect on the fusion proteinagainst the stress environments such as agitation and oxidation, a sugaror a sugar alcohol, a surfactant, and an amino acid were added asexcipients. As a result, sucrose and sorbitol showed protective effecton the fusion protein against oxidation stress, while surfactants suchas tween 20, Tween 80 and poloxamer 188 showed protective effect on thefusion protein against agitation stress, irrespective of the type of thesurfactant. In the cases of further using an amino acid, the protectiveeffect on the fusion protein according to the present invention wasobserved when arginine, glutamine, glycine, or histidine was added,among which glutamate showed an excellent protective effect (Table 4 andTable 5).

By the above method, Tween 80 and sucrose were selected, as showing aprotective effect on the fusion protein, and the protective effect ofthe combination of the selected excipients was examined. As a result, aprotective effect on the fusion protein against agitation or oxidationstress was shown when Tween 80 and sucrose were mixed in thehistidine-acetate basal buffer. Also, the average change of about 1% wasobserved in a variety of stress conditions when sucrose, sorbitol, andTween 80 were mixed in the histidine-acetate basal buffer, verifyingthat such formulation is excellent (Table 7, Table 8 and Table 10).

BEST MODE FOR CARRYING OUT THE PRESENT INVENTION

Hereinafter, the present invention is explained in detail by Examples.The following Examples are intended to further illustrate the presentinvention without limiting its scope.

Example 1: Preparation of Modified Interleukin-7 Fusion Proteins

Prepared was an IL-7 fusion protein in which an oligopeptide and animmunoglobulin Fc region were bound to the N-terminal and theC-terminal, respectively. The sequence of a human IL-7 (SEQ ID NO:1) wasused for IL-7, and the combination sequence of methione (M) and glycine(G) was used for the oligopeptide. The Fc region is a hybrid of the Fcregion of human IgD and the Fc region of human IgG4, which can increasein vivo half-life grately when binding to a bioactive fusion protein,compared to known modified Fc regions of immunoglobulins.

First, gene sequence which encodes the amino acid sequence ofM-IL-7-hyFc, G-IL-7-hyFc, MM-IL-7-hyFc, MG-IL-7-hyFc, GM-IL-7-hyFc,GG-IL-7-hyFc, MMM-IL-7-hyFc, GMM-IL-7-hyFc, MGM-IL-7-hyFc,MMG-IL-7-hyFc, GGM-IL-7-hyFc, GMG-IL-7-hyFc, MGG-IL-7-hyFc,GGG-IL-7-hyFc, MMMM-IL-7-hyFc, GMMM-IL-7-hyFc, MGMM-IL-7-hyFc,MMGM-IL-7-hyFc, MMMG-IL-7-hyFc, GGMM-IL-7-hyFc, MGGM-IL-7-hyFc,MMGG-IL-7-hyFc, GMGM-IL-7-hyFc, MGMG-IL-7-hyFc, GMMG-IL-7-hyFc,GGGM-IL-7-hyFc, MGGG-IL-7-hyFc, GMGG-IL-7-hyFc, GGMG-IL-7-hyFc,GGGG-IL-7-hyFc, MMMMM-IL-7-hyFc, GMMMM-IL-7-hyFc, GGMMM-IL-7-hyFc,GGGMM-IL-7-hyFc, GGGGM-IL-7-hyFc, MGMMM-IL-7-hyFc, MGGMM-IL-7-hyFc,MGGGM-IL-7-hyFc, MGGGG-IL-7-hyFc, MMGMM-IL-7-hyFc, MMGGM-IL-7-hyFc,MMGGG-IL-7-hyFc, MMMGM-IL-7-hyFc, MMMGG-IL-7-hyFc, MMMMG-IL-7-hyFc,MGMGM-IL-7-hyFc, GMGMG-IL-7-hyFc, GMMMG-IL-7-hyFc, GGMGM-IL-7-hyFc,GGMMG-IL-7-hyFc, MGGMG-IL-7-hyFc, MGMGG-IL-7-hyFc, GMMGM-IL-7-hyFc,MGMMG-IL-7-hyFc, GMGGM-IL-7-hyFc, MMGMG-IL-7-hyFc, GMMGG-IL-7-hyFc,GMGGG-IL-7-hyFc, GGMGG-IL-7-hyFc, GGGMG-IL-7-hyFc or GGGGG-IL-7-hyFc wasinserted into pAD15 expression vector. In order to insert a fusionprotein gene into an expression vector pAD15, EcoRI site was generatedat the 5′-end of the fusion protein gene sequence, and XbaI site wasgenerated at the 3′-end of the hyFc gene sequence. Expression vectorpAD15 was obtained from RcCMV backbone (available at Invitrogen,Carlsbad). The pAD15 contains a cytomegalovirus (CMV)-derived promoter,fetal bovine growth hormone-derived poly (A) sequence, rabbit betaglobin-derived gIVS (globin intervening sequence) (Mol Cell Biol. 1988,8: 4395), and other factors. The pAD15 vector was prepared by modifyingvarious parts of RcCMV vector (Invitrogen). First, neomycin resistancesite was removed by XhoI enzyme, and gIVS was added to the 3′-end of theCMV promoter region. In addition, mouse dihydrofolate reductase (DHFR)gene (GeneBank Accession No. NM 010049) was added to the 5′-end of theCMV promoter. In order to prepare the connection site between the 3′-endof IL-7 and the 5′-end of hyFc in the frame, NheI site was generated atthe 3′-end of IL-7 coding sequence and the 5′-end of hyFc codingsequence. The final expression vector was prepared through a subcloningprocess using each restriction enzyme site. For more informationregarding the preparation of the modified Fc region, U.S. Pat. No.7,867,491 may be referred to (indicated as “hybrid (Fc) protein”). ThehyFc protein of the present Example contains 9 amino acids of theC-terminal of IgD CH1 domain (90-98), 30 amino acids of IgD hinge region(133-162), 8 amino acids of the N-terminal of IgD CH2 domain (SHTQPLGV163-170), 100 amino acids of IgG4 CH2 domain (121-220), and 107 aminoacids of IgG4 CH3 domain (221-327).

Regarding the hyFc protein composed of a modified Fc region, the presentinvention takes reference to every preparation method for hFc-2, hFc-3,hFc-4, hFc-5 and hFc-6 described in the Example of the Korean PatentPublication No. 10-0897938. The hyFc protein used in the Examples of thepresent invention is a hybrid protein identical to hFc-5 described inthe Korean Patent Publication No. 10-0897938.

PCR was carried out under the condition shown in Table 1. Denaturation,annealing and extension processes were carried out for 15 seconds at 98°C., for 30 seconds at 55° C., and for 60 seconds at 72° C.,respectively.

TABLE 1 pAD15 template comprising the gene of a modified IL-7 2 μlfusion protein Forward primer (5′-accgaattcatgttccacgtgagcttcag-3′) 1.5μl   Reverse primer (5′-ggttctagattagtgctccttggtgcccatc-3′) 1.5 μl   5 ×HF buffer 20 μl  2.5 mM dNTP 8 μl DMSO 3 μl Phusion (Thermo Scientific)1 μl Tertiary distilled water 64 μl 

The prepared expression vector, which is M-IL-7-hyFc, G-IL-7-hyFc,MM-IL-7-hyFc, MG-IL-7-hyFc, GM-IL-7-hyFc, GG-IL-7-hyFc, MMM-IL-7-hyFc,GMM-IL-7-hyFc, MGM-IL-7-hyFc, MMG-IL-7-hyFc, GGM-IL-7-hyFc,GMG-IL-7-hyFc, MGG-IL-7-hyFc, GGG-IL-7-hyFc, MMMM-IL-7-hyFc,GMMM-IL-7-hyFc, MGMM-IL-7-hyFc, MMGM-IL-7-hyFc, MMMG-IL-7-hyFc,GGMM-IL-7-hyFc, MGGM-IL-7-hyFc, MMGG-IL-7-hyFc, GMGM-IL-7-hyFc,MGMG-IL-7-hyFc, GMMG-IL-7-hyFc, GGGM-IL-7-hyFc, MGGG-IL-7-hyFc,GMGG-IL-7-hyFc, GGMG-IL-7-hyFc, GGGG-IL-7-hyFc, MMMMM-IL-7-hyFc,GMMMM-IL-7-hyFc, GGMMM-IL-7-hyFc, GGGMM-IL-7-hyFc, GGGGM-IL-7-hyFc,MGMMM-IL-7-hyFc, MGGMM-IL-7-hyFc, MGGGM-IL-7-hyFc, MGGGG-IL-7-hyFc,MMGMM-IL-7-hyFc, MMGGM-IL-7-hyFc, MMGGG-IL-7-hyFc, MMMGM-IL-7-hyFc,MMMGG-IL-7-hyFc, MMMMG-IL-7-hyFc, MGMGM-IL-7-hyFc, GMGMG-IL-7-hyFc,GMMMG-IL-7-hyFc, GGMGM-IL-7-hyFc, GGMMG-IL-7-hyFc, MGGMG-IL-7-hyFc,MGMGG-IL-7-hyFc, GMMGM-IL-7-hyFc, MGMMG-IL-7-hyFc, GMGGM-IL-7-hyFc,MMGMG-IL-7-hyFc, GMMGG-IL-7-hyFc, GMGGG-IL-7-hyFc, GGMGG-IL-7-hyFc,GGGMG-IL-7-hyFc or GGGGG-IL-7-hyFc, was transfected into CHO DG44 cells(from Dr. Chasin, Columbia University, U.S.A.) by an electroporationmethod. After 48 hours, HT selection was carried out using 10% dFBS+MEMα medium without HT, and MTX amplification was carried out using HTselected clones to amplify the productivity. The obtained single cellswere used for the preparation of a modified IL-7 fusion protein aftertesting the long-term stability thereof.

Example 2: Purification of Modified IL-7 Fusion Protein

After obtaining the culture solution containing the modified IL-7 fusionprotein prepared by the method of Example 1, the productivity wasmeasured by performing size-exclusion (SE) HPLC according to theconditions shown in Table 2 below. SE-HPLC was carried out for 40minutes using TSK-GEL G3000WxL column.

First, each of the sample solutions of the modified IL-7 fusion proteinwas diluted with a buffer to a concentration of 1 mg/ml. Each diluentwas filtered with 1 ml syringe and 0.2 μm filter and then put into aninsert. The insert was inserted into a vial and the cap was kept closed.Then, each of the test solutions was injected into SE-HPLC system in anamount of 20 μl. The purity and relative productivity were measuredbased on the peak value.

As a result, the modified IL-7 fusion protein with the structure shownin FIG. 1, which has high productivity and purity, was purified(preparation and purification of the modified IL-7 fusion protein werecarried out according to Korea Patent Application No. 10-2015-0082793).

Experimental Example 1: Selection of Basal Buffer

1.1. Selection Based on Thermodynamic Property

To establish a formulation of the modified IL-7 fusion protein purifiedin Example 2 above, the thermodynamic property of the fusion proteins inthree types of buffer was examined by differential scanning calorimetry(DSC) method.

First, the modified IL-7 fusion protein purified in Example 2 wassubjected to dialysis at 4° C. using 20 mM histidine-acetate buffer orsodium citrate buffer with a pH of 5.0, 5.5, or 6.0, or tris buffer witha pH of 8.0.

Meanwhile, DSC was carried out using Microcal VP-DSC (Northampton,U.S.A.) having twin cells with the volume of 0.5147 cm³ regarding thesamples and a control solution. The scanning was conducted at the speedof 1.25° C./min at the temperature from 15° C. to 110° C. Beforemeasuring the values, every solution was agitated in a vacuum state toremove air. Then, the standard value for a solution without the fusionprotein was first determined, and the temperature record of a modifiedIL-7 fusion protein was shown by the values determined by subtractingthe standard value from the measured values.

The resulting graph was shown by using Microcal LLC DSC (Northampton,U.S.A.) which was connected with Origin 7.0 software package providedwith the device. In addition, from the results, the transitiontemperature (Tm) and enthalpy (ΔH) values of the fusion protein wereobtained by using Origin 7.0 software.

As shown in FIG. 2, in the case of MGM-IL-7-hyFc fusion protein, Tm 1and Tm 2, the transition temperatures of IL-7 and hyFc, respectively,and two peaks representing the fusion protein structure unfolded by heatwere identified in the typical DSC thermogram regarding the three typesof buffers. These results indicate that each of IL-7 and hyFc exists inan unfolded state due to heat energy absorption, and they arestructurally stable.

In general, hyFc is a design platform for the preparation of along-acting protein in which IgD (hinge-C_(H)2) and IgG4 (C_(H)2-C_(H)3)are hybridized, and has single or double transitions from IgG4 accordingto various environmental factors. However, the result of the presentexperiment showed that apparently hyFc had a single transitional peak.

In addition, as shown in Table 2 below, Tm 1 and Tm 2 respectivelyincreased from 58.89° C. to 62.92° C. and from 66.93° C. to 69.91° C.,as the pH of the histidine-acetate buffer increased from 5.0 to 6.0.Moreover, ΔH also increased along with Tm. These results indicate thatas pH increases, higher temperature and more heat energy are necessaryfor unfolding the structure of IL-7 and hyFc. That is, conformationalstability of IL-7 and hyFc protein might increase as pH increases.

Even when sodium citrate with the pH of 5.0, 5.5, or 6.0 was used as abasal buffer, Tm 1 and Tm 2 increased as the pH increased, which resultswere identical to the results obtained by using a histidine-acetatebuffer. It should be noted that Tm 1 and Tm 2, respectively, increasedfrom 57.23° C. to 65.12° C. and from 66.74° C. to 71.34° C. as the pHincreased from 5.0 to 6.0.

Regarding ΔH value when using sodium citrate as a basal buffer, ΔHvalues of Tm 1 and Tm 2 were the lowest when pH was 5.0. This resultindicates that the suitable pH of a basal buffer for stably sustainingthe structure of the fusion protein is 5.5 or 6.0.

When tris buffer of pH 8.0 was used, Tm 1, Tm 2 and ΔH value of Tm 1were higher than those obtained with other buffers. This result wasconsistent with the aforementioned results which showed that theconformational stability of a modified IL-7 fusion protein increased aspH increased. In the case of tris buffer with a high pH, Tm 2 valueincreased compared to other buffers with a pH of 6.0, but the ΔH valueof Tm 2 decreased, indicating that it might have bigger influence on theconformational stability of IL-7 than on the stability of hyFc.

TABLE 2 Transition Buffer/pH temperature Enthalpy (MGM-IL-7-hyFc (° C.)(kJ/mol) concentration, mg/ml) Tm 1 Tm 2 ΔH1 ΔH2 Histidine-acetate pH5.0 (8.6) 58.89 66.93 263.47 102.91 pH 5.5 (8.8) 61.11 68.72 290.68104.17 pH 6.0 (8.6) 62.92 69.91 332.06 115.41 Sodium citrate pH 5.0(9.3) 57.23 66.74 242.15 110.48 pH 5.5 (9.6) 62.17 70.28 305.27 124.77pH 6.0 (9.8) 65.12 71.34 292.10 126.19 Tris pH 8.0 (7.8) 71.53 66.06384.27 104.58

1.2. Selection by Dynamic Light Scattering (DLS) Analysis

DLS analysis was carried out in order to evaluate the electrostaticinteraction between proteins, and Z-average size, polydispersity index(PDI) and zeta potential, and the effect of pH and a buffer on theaggregates formation. The particle size of the protein and zetapotential were measured using Zetasizer Nano ZS90 (Malvern Instruments,UK).

Z-average size refers to the average of elements such as a fragment, amonomer and an aggregate, which is very sensitive to even small changessuch as the presence of a small aggregate portion. If the measured PDIvalue is lower than 0.05, it is shown as a monodisperse standard, and ifthe value is higher than 0.7, it indicates that the sample has a widesize distribution. Zeta potential is an electrical repulsion of proteinssurrounding a hydrodynamic surface. If the absolute value of the zetapotential is high, the protein is less subject to aggregation andinstability in a solution.

First, for the DLS analysis, a modified IL-7 fusion protein sample wasequilibrated at the measurement temperature of 10° C. Using 1 ml of thesample, particle size was measured 5 times in a disposable sizingcuvette, and zeta potential was measured 3 times in a disposablecapillary cell with the angle fixed to 90°. Intensity of the peak,average particle size, PDI and the zeta potential were calculated usingZetasizer software version 7.11 (Malvern Instruments, UK), which wasprovided with the device.

The protein size distribution represented by the intensity of proteinpeaks when using histidine-acetate or sodium citrate of pH 5.0, or trisof pH 8.0 as a basal buffer was shown in FIG. 3. The Z-average size, PDIand zeta potential of the three buffers in different pH conditions wereshown in Table 3.

As shown in FIG. 3, when histidine-acetate or sodium citrate of pH 5.0was used as a basal buffer, the resulting graph showed a single narrowpeak throughout 5 consecutive measurements. Meanwhile, when tris bufferof pH 8.0 was used, a new peak was identified, which suggestedaggregation at 100 to 1,000 nm.

As shown in Table 3, when histidine-acetate was used as a basal buffer,new aggregation appeared as pH increased from 5.0 to 6.0, and theZ-average size and PDI, respectively, increased from 10.43 nm to 10.64nm, and 0.02 to 0.07. In addition, as pH increased, the absolute valueof zeta potential decreased from 4.42 to 1.28.

The aggregation phenomenon according to the pH change as described abovewas also observed when sodium citrate was used as a basal buffer: thatis, Z-average size and PDI increased and zeta potential decreased in asodium citrate basal buffer.

However, Z-average size and PDI in a tris basal buffer highly increasedto 12.38 nm and 0.34, respectively, as compared to those in otherbuffers. In addition, protein aggregation took place in the tris basalbuffer. Also, even though aggregation took place, the absolute value ofzeta potential in the tris buffer was 20.27, which was higher than thosein other buffers.

Therefore, in order to reduce the aggregation of the modified IL-7fusion protein of the present invention, histidine-acetate or sodiumcitrate may be suitable as a basal buffer, preferably in the pHcondition with low zeta potential. In a high pH condition, the buffercan be used in combination with protein aggregation inhibitor.

TABLE 3 Buffer/pH (MGM-IL-7-hyFc Z-average size Absolute value ofconcentration, mg/ml) (d.nm*) PDI zeta potential (mV) Histidine-acetatepH 5.0 (8.6) 10.43 ± 0.06 0.02 ± 0.01 4.42 ± 0.32 pH 5.5 (8.8) 10.58 ±0.09 0.03 ± 0.02 1.11 ± 0.43 pH 6.0 (8.6) 10.64 ± 0.24 0.07 ± 0.05 1.28± 0.47 Sodium citrate pH 5.0 (9.3) 10.73 ± 0.09 0.02 ± 0.01 5.18 ± 0.74pH 5.5 (9.6) 10.82 ± 0.10 0.03 ± 0.02 3.38 ± 0.57 pH 6.0 (9.8) 11.03 ±0.17 0.08 ± 0.03 2.46 ± 0.42 Tris pH 8.0 (7.8) 12.38 ± 0.84 0.34 ± 0.0520.27 ± 0.42  *d.nm: diameter in nanometer

Experimental Example 2: Selection of Excipients

2.1. Selection According to the Protective Effect on Stress Conditions

In order to examine the protective effect of excipients against stressenvironments such as agitation and oxidation, various excipientsincluding a surfactant with a concentration of 5 w/v %, a sugar with aconcentration of 5 w/v % and an amino acid with a concentration of 50mM, were subjected to a size-exclusion chromatography. In the controlgroup, only modified IL-7 fusion protein was added to a basal buffer.For the basal buffer, histidine-acetate or sodium citrate basal bufferof pH 5.0 was used.

Specifically, the modified IL-7 fusion proteins were dialyzed at 4° C.with the solutions prepared by adding various types of excipients tohistidine-acetate or sodium citrate basal buffers of pH 5.0. For theagitation stress environment, 0.5 ml of the modified IL-7 fusion proteinas dialyzed was put into a microtube, which was then put into a box andagitated for 2 hours at room temperature using Vortex-Genie 2(Scientific Industries, Inc., U.S.A.) at strength 7.

Meanwhile, for the oxidation stress environment, 0.5 ml of the dialyzedMGM-IL-7-hyFc fusion protein was added with hydrogen peroxide to thefinal concentration of 1.0% (v/v) and stored for 20 hours at roomtemperature. Size-exclusion chromatography was carried out using highperformance liquid chromatography (HPLC) system (Waters e2695, U.S.A.)at the UV wavelength of 214 nm of absorption spectrum.

<HPLC Condition>

Column: TSKgel G3000SWXL SEC column (300×7.8 mm) (TOSOH Bioscience,U.S.A.)

Mobile phase: solution containing 100 mM NaCl and 10% acetonitrile in 50mM sodium phosphate (pH 6.8)

Flow rate: 0.5 ml/min

As a result, several elements including water-soluble aggregation,monomers and fragments were observed, and the content of each elementwas calculated by the mathematical equation 1.

% content of specific element=C _(t) /C _(s)×100  [Equation 1]

Herein, Ct refers to the area of each element (water-solubleaggregation, monomer and fragment), and Cs refers the sum of the peaksof the elements when a sample is obtained.

As a result, as shown in Tables 4 and 5, the monomer content in a sodiumcitrate buffer in the control group was 96.34%, which decreased to78.18% by agitation and 48.16% by oxidation.

It was found that MGM-IL-7-hyFc fusion protein was protected from theagitation stress by adding widely-known non-ionic surfactant such asTween or poloxamer to a histidine-acetate or sodium citrate buffer. Suchnon-ionic surfactants have an excellent effect of protecting the proteinfrom uncoiling in a hydrophobic interface. When Twin 20, Twin 80 orpoloxamer 188 were added to a buffer, the monomer content did notsignificantly decrease. However, the protective effects of thesesurfactants were substantially similar, indicating that the surfactantsprotected the fusion protein of the present invention in a hydrophobicenvironment which constantly changes due to agitation.

Meanwhile, the surfactants did not show any protective effect onMGM-IL-7-hyFc fusion protein against oxidation stress, but rather causedmore reduction of the monomer content than the control group. Moreover,Twin 20 and Twin 80 are known to oxidate a protein. Poloxamer 188 alsomade the fusion protein of the present invention unstable in theoxidation condition, as Twin 20 and Twin 80 did.

A sugar or a sugar alcohol such as sucrose and sorbitol is often used asa pharmaceutical excipient for stabilizing MGM-IL-7-hyFc fusion protein.However, sucrose and sorbitol caused lower monomer content than thecontrol group, which indicated that they do not have a protective effecton the fusion protein of the present invention against agitation stress.

After agitation in the sodium citrate buffer, based on the referencevalue not exposed to a stress, the monomer content decreased by 18.16%in the control group, while the monomer contents decreased by 39.29% and53.51% when sucrose and sorbitol, respectively, were added. Meanwhile,after agitation in the histidine-acetate buffer, based on the referencevalue not exposed to a stress, the monomer content decreased by 2.23% inthe control group, while the monomer contents decreased by 11.53% and13.77% when sucrose and sorbitol, respectively, were added.

It is known that the addition of a sugar and a sugar alcohol to aformulation stimulates aggregates formation of IgG during agitation.When sucrose or sorbitol were added to a formulation, the aggregatesformation increased during agitation, which led to the decrease in thestability of MGM-IL-7-hyFc fusion protein, irrespective of the kind of abasal buffer.

Meanwhile, sucrose and sorbitol increased the stability of a fusionprotein under the oxidation stress. In a sodium citrate buffer, themonomer content decreased by 48.18% in the control group based on thereference value not exposed to a stress, while the monomer contentsdecreased by 2.55% and 2.79% when sucrose and sorbitol, respectively,were added. Similarly, in a histidine-acetate buffer, the monomercontent decreased by 35.36% in the control group based on the referencevalue not exposed to a stress, while the monomer contents decreased by1.68% and 1.47% when sucrose and sorbitol, respectively, were added.This result indicates that sucrose and sorbitol have a protective effecton protein from oxidation. The two substances are reported to be able tostabilize a protein by preferential exclusion from a surface of aprotein.

Next, with regard to the effect of an amino acid on the stability ofMGM-IL-7-hyFc fusion protein under a stress condition, the protectiveeffect against agitation was very low when 50 mM arginine, glutamate,glycine and histidine amino acids were respectively added to sodiumcitrate buffers. The monomer content decreased by 18.16% in the controlgroup based on the reference value not exposed to a stress, while themonomer contents decreased by 49.71%, 64.75%, 30.96% and 46.75% whenarginine, glutamate, glycine and histidine were, respectively, added.Meanwhile, arginine and glutamate were found to make a fusion protein ofthe present invention unstable when histidine-acetate buffer was used.

Meanwhile, when glycine was added to a histidine-acetate buffer, themonomer content of the MGM-IL-7-hyFc fusion protein did not decrease ascompared to the reference value not exposed to a stress. The result wasconflicting to the result from a sodium citrate buffer, suggesting thatthe stability of MGM-IL-7-hyFc fusion protein can change depending onthe environmental factors including a buffer and an excipient.

Moreover, the protective effect against oxidation stress was observedwhen arginine and glycine were respectively added to sodium citratebuffers, but such effect was not observed when they were added tohistidine-acetate buffers. However, with regard to the oxidation stress,when arginine and glycine were respectively added to sodium citratebuffers, the samples showed decreases of 30.66% and 3.21% respectivelyin the monomer contents based on the reference value not exposed to astress, which were smaller than in the control group (decreased by48.18%). Meanwhile, when arginine and glycine were added tohistidine-acetate buffers, the monomer contents decreased significantlyby 39.34% and 37.90% respectively based on the reference value notexposed to a stress, compared to the control group (decreased by35.36%). Especially, in both types of buffers, the addition of glycineshowed a protective effect on the fusion protein of the presentinvention against agitation and oxidation stresses depending on buffertypes, and the addition of glutamate resulted in an excellent protectiveeffect on the fusion protein against oxidation stress.

TABLE 4 Sodium citrate pH 5.0 [MGM-IL-7-hyFc concentration: 9.5 mg/ml(Final concentration 3.0 mg/ml)] Aggregate (%) Monomer (%) Fragment (%)Excipient Reference* Agitation Oxidation Reference* Agitation OxidationReference* Agitation Oxidation Control group 0.81 20.58 47.98 96.3478.18 48.16 2.85 1.24 3.86 Sugar & Sucrose 0.69 41.78 0.65 95.65 56.3693.10 3.66 1.86 6.25 Sugar Sorbitol 0.57 55.79 0.98 96.08 42.57 93.293.34 1.64 5.74 alcohol Surfactant Tween 20 0.57 0.84 48.13 95.74 95.7748.02 3.69 3.39 3.85 Tween 80 0.69 0.64 48.02 95.76 95.80 47.34 3.553.56 4.63 Poloxamer 0.69 0.74 50.17 95.71 95.68 46.45 3.60 3.58 3.38 188Amino Arginine 0.75 52.65 31.39 95.62 45.91 64.96 3.36 1.44 3.65 acidGlutamate 0.55 67.66 0.81 95.60 30.85 93.73 3.85 1.48 5.46 Glycine 0.6934.09 2.44 95.76 64.80 92.55 3.55 1.11 5.01 Histidine 0.77 50.34 22.3795.70 48.95 73.53 3.53 0.72 4.10 *Note: Not exposed to a stress

TABLE 5 Histidine-acetate pH 5.0 [MGM-IL-7-hyFc concentration: 9.5 mg/ml(Final concentration 3.0 mg/ml)] Aggregate (%) Monomer (%) Fragment (%)Excipient Reference* Agitation Oxidation Reference* Agitation OxidationReference* Agitation Oxidation Control group 0.45 4.52 35.03 95.39 93.1660.03 4.16 2.33 4.94 Sugar & Sucrose 0.42 13.71 0.52 95.62 84.09 93.943.96 2.20 5.54 Sugar Sorbitol 0.46 15.80 0.37 95.74 81.97 94.27 3.802.23 5.36 alcohol Surfactant Tween 20 0.43 0.55 39.36 95.32 95.73 56.594.25 3.72 4.06 Tween 80 0.36 0.60 40.93 96.00 95.30 55.29 3.64 4.10 3.79Poloxamer 0.50 0.64 41.89 95.77 95.87 54.37 3.73 3.49 3.75 188 AminoArginine 0.51 29.11 39.35 96.04 69.26 56.70 3.45 1.63 3.95 acidGlutamate 0.42 56.11 0.77 96.22 39.40 93.87 3.36 1.50 5.36 Glycine 0.532.93 38.42 95.55 94.83 57.65 3.92 2.24 3.93 *Note: Not exposed to astress

2.2. Selection by DLS Analysis

To examine the effect of various excipients including a surfactant, asugar and an amino acid on the stability of a modified IL-7 fusionprotein, DLS analysis was conducted by the method described inExperimental Example 1.2. The measurements of a particle size and zetapotential were carried out three times, respectively.

Z-average size of MGM-IL-7-hyFc fusion proteins which were exposed tothe agitation and oxidation stress in sodium citrate buffer with pH 5.0was shown in FIG. 4 and Table 6.

TABLE 6 Sodium citrate pH 5.0 Reference* Agitation Oxidation Z-averageZ-average Z-average Excipient size (d · nm) PDI size (d · nm) PDI size(d · nm) PDI) Control group 11.29 0.07 19.40 0.19 177.10  0.85 Sugar &Sucrose 11.80 0.17 — — 13.07 0.28 Sugar alcohol Sorbitol 11.89 0.14 — —14.11 0.32 Surfactant Tween 20 10.61 0.07 10.77 0.11 — — Tween 80 10.690.03 10.78 0.07 — — Poloxamer 188 11.01 0.05 11.32 0.11 — — Amino acidGlutamate 11.09 0.06 — — 11.26 0.06 Glycine 11.65 0.13 — — 50.71 0.58*Note: Not exposed to a stress

As shown in FIG. 4a , the control group showed only a single narrow peakin the particle size measurement which was conducted three times.However, the agitation control group, which was exposed to an agitationstress, showed another peak at the size of 100 nm. In this controlgroup, Z-average size increased from 11.29 nm to 19.40 nm, and PDI alsoincreased from 0.07 to 0.19, indicating that an aggregation formationtook place. Meanwhile, when Twin 20, Twin 80 and poloxamer 188 wereadded, Z-average size increased by 0.16, 0.09, 0.31 nm, respectively,and PDI increased by 0.04, 0.04 and 0.06, respectively. The increase inZ-average size and PDI implies that the aggregation is induced byagitation. A smaller increase was observed by the addition of asurfactant, indicating that the surfactants had a protein protectiveeffect from aggregates formation. As shown in FIG. 4b , an aggregatepeak was not observed when a surfactant was added.

The oxidation control group, which was exposed to an oxidation stress,showed an additional peak at the size of 1,000 nm, while Z-average sizeand PDI increased from 11.29 nm to 177.10 nm, and 0.07 to 0.85 nm,respectively. This result indicates that an aggregate is formed byoxidation, and the formed aggregate is bigger than that formed byagitation. As shown in FIG. 4c , an aggregate peak was also observedwhen sucrose, sorbitol and glycine were respectively added, butaggregates were not formed when glutamate was added. In the case ofglutamate-added sample, Z-average size increased by 0.17 nm and PDI didnot change. This result indicates that when glutamate is added to sodiumcitrate buffer with pH 5.0, it shows an excellent protective effect onMGM-IL-7-hyFc fusion protein against oxidation stress. Meanwhile, whensucrose, sorbitol and glycine were added, Z-average size increased by1.27 nm, 2.22 nm and 39.06 nm, respectively, and PDI increased by 0.11,0.18 and 0.45, respectively, which were considered to be small whencompared to the samples in which other excipients were added. Thisresult indicates that sucrose, sorbitol and glycine have a protectiveeffect on the fusion protein of the present invention against oxidationstress, which is consistent with the result of Experimental Example 2.1.

Meanwhile, the Z-average size of MGM-IL-7-hyFc fusion proteins whichwere exposed to agitation and oxidation stress in histidine-acetatebuffer with pH 5.0 was shown in FIG. 5 and Table 7.

TABLE 7 Histidine-acetate pH 5.0 Reference* Agitation OxidationZ-average Z-average Z-average Excipient size (d · nm) PDI size (d · nm)PDI size (d · nm) PDI Control group 10.50 0.02 11.70 0.10 17.43 0.37Sugar & Sucrose 11.21 0.17 — — 12.47 0.22 Sugar alcohol Sorbitol 12.020.13 — — 11.61 0.09 Surfactant Tween 20 10.66 0.10 10.79 0.12 — — Tween80 10.40 0.03 10.50 0.03 — — Poloxamer 188 10.83 0.04 10.74 0.07 — —Amino acid Glutamate 10.90 0.04 — — 11.31 0.06 Glycine 10.68 0.03 15.660.41 — — *Note: Not exposed to a stress

As shown in FIG. 5a , the control group and the agitation control groupshowed a single narrow peak, which is a different result from theagitation control group with the sodium citrate buffer. However, asshown in FIG. 5b , an aggregate peak was observed when glycine wasadded, but an aggregate peak was not observed and Z-average size and PDIdid not change when surfactants such as Twin 20, Twin 80 and poloxamer188 were added. This result indicates that an aggregates formation cantake place by agitation when glycine is added to a MGM-IL-7-hyFc fusionprotein formulation.

As shown in FIG. 5c , when sucrose was added, Z-average size and PDI,respectively, increased from 11.21 nm to 12.47 nm, and 0.17 to 0.22 byoxidation. Both of the changes were smaller than the control group,indicating that sucrose has a protein protective effect againstoxidation stress, but cause the problem of aggregates formation.Meanwhile, when sorbitol was added, Z-average size and PDI respectivelydecreased from 12.02 nm to 11.61 nm, and 0.13 to 0.09, while noaggregate peak was found. The decreases in Z-average size and PDIsuggest the increase of fragments, even if not significant. Whenglutamate was added, Z-average size and PDI increased slightly, whileneither aggregates nor fragments were found.

Therefore, it was found that the surfactants such as Twin 20, Twin 80and poloxamer 188 have a protective effect on the fusion protein of thepresent invention against oxidation or agitation stress; a sugar or asugar alcohol such as sucrose and sorbitol exhibits a protective effectagainst oxidation stress; and amino acids show different effectsaccording to the basal buffer, but overall, glutamate shows a highprotective effect.

Experimental Example 3: Combination of Excipients

From the results of Experimental Example 2, Tween 80 and sucrose wereselected as the excipients showing a protective effect on the modifiedIL-7 fusion protein against agitation and oxidation stresses,respectively. For experiment for the combination of these twoexcipients, Design of Experiment software (Design-Expert, Stat-EaseInc., U.S.A.) was used. The two excipients were combined as shown inTable 8, and the effect thereof on stability of the modified IL-7 fusionprotein was examined. Stress condition in the present ExperimentalExample was the same as Experimental Example 2.1., and the finalconcentration of the modified IL-7 fusion protein in the formulation wasadjusted to 3 to 100 mg/ml.

TABLE 8 Excipient (Addition concentration w/v %) Tween 80 Sucrose 1 0.000 2 0.05 0 3 0.10 0 4 0.00 2.5 5 0.05 2.5 6 0.10 2.5 7 0.00 5 8 0.05 5 90.10 5 10 0.05 2.5

To examine the protective effect against a stress condition,size-exclusion chromatography was carried out [at 214 nm of UVwavelength of absorption spectrum using high performance liquidchromatography (HPLC) system (Waters e2695, U.S.A.)], under the sameHPLC condition as Experimental Example 2.1.

First, an experiment was conducted regarding a formulation comprisingthe modified IL-7 fusion protein in a concentration of 3 mg/ml. As aresult, as shown in Table 9, the control group with sodium citratebuffer showed a monomer content of 95.65%, which decreased to 74.77% byagitation, and to 49.81% by oxidation. The samples in which Tween 80 wasadded showed a protective effect against agitation stress, while thosein which sucrose was added showed a protective effect against oxidationstress. The samples in which Tween 80 and sucrose were added incombination showed the protective effect against agitation stress andoxidation stress.

TABLE 9 Final Basal concentration SE-HPLC (%) Buffer Sample Excipient(w/v %) Reference* Agitation Oxidation Sodium 1 Tween 80 Sucrose 0.00 095.65 74.77 49.81 citrate 2 Tween 80 Sucrose 0.05 0 95.06 95.39 48.88 3Tween 80 Sucrose 0.10 0 95.39 95.28 50.51 4 Tween 80 Sucrose 0.00 2.594.42 71.26 92.24 5 Tween 80 Sucrose 0.05 2.5 95.08 95.19 91.93 6 Tween80 Sucrose 0.10 2.5 95.06 95.85 91.68 7 Tween 80 Sucrose 0.00 5 95.2971.70 92.67 8 Tween 80 Sucrose 0.05 5 95.51 95.02 91.96 9 Tween 80Sucrose 0.10 5 95.42 95.27 92.49 10 Tween 80 Sucrose 0.05 2.5 94.9695.30 91.71 *Note: Not exposed to a stress

Meanwhile, as shown in Table 10, the control group withhistidine-acetate buffer showed a monomer content of 95.46%, whichdecreased to 93.60% by agitation, and to 50.63% by oxidation. Ascompared to the sodium citrate buffer, the histidine-acetate buffershowed more excellent protective effect against agitation stress, andthe protective effects of the added excipients was similar to those inthe samples with sodium citrate.

TABLE 10 Final Basal concentration SE-HPLC (%) buffer Sample Excipient(w/v %) Reference* Agitation Oxidation Histidine- 1 Tween 80 Sucrose0.00 0 95.46 93.60 50.63 acetate 2 Tween 80 Sucrose 0.05 0 95.77 94.6250.68 3 Tween 80 Sucrose 0.10 0 95.35 95.30 50.59 4 Tween 80 Sucrose0.00 2.5 95.49 94.93 93.23 5 Tween 80 Sucrose 0.05 2.5 95.52 94.36 92.326 Tween 80 Sucrose 0.10 2.5 95.75 94.82 91.43 7 Tween 80 Sucrose 0.00 595.34 94.86 93.66 8 Tween 80 Sucrose 0.05 5 95.29 94.90 93.36 9 Tween 80Sucrose 0.10 5 95.38 94.42 93.58 10 Tween 80 Sucrose 0.05 2.5 94.9094.90 92.12

From the results, the condition of the excipients was selected. Toacquire the proper osmotic strength of an injection, sorbitol andmannitol were further added, and agitation stress and oxidation stressexperiments were conducted, and additional experiment under thecondition of adding 1.5 w/v % sorbitol was also conducted.

As a candidate formulation for each of sodium citrate buffer andhistidine-acetate buffer, 5 w/v % sucrose, 1.5 w/v % sorbitol, and 0.05w/v % Tween 80 were added to the formulations for MGM-IL-7-hyFc fusionprotein. Using the above formulations, freeze/thaw, temperature stressand oxidation stress experiments were conducted. The stress conditionsare shown in Table 11, and the analysis result is shown in Table 12.

TABLE 11 Stress condition Condition Stress method Control group −80° C.Freeze/thaw −80° C. → Room temperature (5 times) Temperature 37° C. (39h, 3 day), 50° C. (20 h) Oxidation 0.1% or 1% H₂O₂, room temperature 20h

TABLE 12 Osmotic pH pressure SE-HPLC (%) Before Before Before stressstress stress 37° C., 50° C., 0.1% Basal buffer exposure exposureexposure Freeze/thaw 39 h 20 h H₂O₂ 1% H₂O₂ 20 mM Sodium citrate, 5.02303 95.76 95.77 94.83 93.81 95.71 92.86 5w/v % Sucrose, 1.5 w/v %Sorbitol, 0.05 w/v % Tween 80, pH 5.0 20 mM Histidine-acetate, 4.98 30095.59 95.64 95.41 95.47 95.95 93.83 5 w/v % Sucrose, 1.5 w/v % Sorbitol,0.05 w/v % Tween 80, pH 5.0

As a result of the stress test using the candidate formulations, asshown in Table 12, in this case where a sodium citrate buffer is used,the control group showed a monomer content of 95.76%, and about 3% ofchange by stress was observed.

In the case where a histidine-acetate buffer is used, the control groupshowed a monomer content of 95.59%, and about 1% of change by stress wasobserved, showing a slightly better protective effect compared to thesodium citrate buffer.

The effect of the candidate formulations on the modified IL-7 fusionprotein of various concentrations was examined. First, a formulationcomprising 3 to 100 mg/ml modified IL-7 fusion protein, 20 mM sodiumcitrate, and 5 w/v % sucrose was prepared. Then, 1 to 2 w/v % sorbitolor mannitol as a sugar alcohol, and 0.05 w/v % Tween 80 or poloxamer asa surfactant were added thereto, and the final pH was adjusted to 5.0.The result indicated that, 3 to 100 mg/ml of modified IL-7 fusionprotein was well protected under the various combination conditions.

1. A pharmaceutical formulation comprising: (a) a modified IL-7 fusionprotein; (b) a basal buffer with a concentration of 10 to 50 mM; (c) asugar with a concentration of 2.5 to 5 w/v %; and (d) a surfactant witha concentration of 0.05 to 6 w/v %.
 2. The pharmaceutical formulation ofclaim 1, wherein the modified IL-7 fusion protein comprises anoligopeptide comprising 1 to 5 amino acids selected from M and G; IL-7;and a Fc region of an immunoglobulin.
 3. The pharmaceutical formulationof claim 2, wherein the oligopeptide comprising 1 to 5 amino acidsselected from M and G is one selected from the group consisting of M, G,MM, MG, GM, GG, MMM, GMM, MGM, MMG, GGM, GMG, MGG, GGG, MMMM (SEQ IDNO:9), GMMM (SEQ ID NO:10), MGMM (SEQ ID NO:11), MMGM (SEQ ID NO:12),MMMG (SEQ ID NO:13), GGMM (SEQ ID NO:14), MGGM (SEQ ID NO:15), MMGG (SEQID NO:16), GMGM (SEQ ID NO:17), MGMG (SEQ ID NO:18), GMMG (SEQ IDNO:19), GGGM (SEQ ID NO:20), MGGG (SEQ ID NO:21), GMGG (SEQ ID NO:22),GGMG (SEQ ID NO:23), GGGG (SEQ ID NO:24), MMMMM (SEQ ID NO:25), GMMMM(SEQ ID NO:26), GGMMM (SEQ ID NO:27), GGGMM (SEQ ID NO:28), GGGGM (SEQID NO:29), MGMMM (SEQ ID NO:30), MGGMM (SEQ ID NO:31), MGGGM (SEQ IDNO:32), MGGGG (SEQ ID NO:33), MMGMM (SEQ ID NO:34), MMGGM (SEQ IDNO:35), MMGGG (SEQ ID NO:36), MMMGM (SEQ ID NO:37), MMMGG (SEQ IDNO:38), MMMMG (SEQ ID NO:39), MGGGM (SEQ ID NO:40), MGMGM (SEQ IDNO:41), GMGMG (SEQ ID NO:42), GMMMG (SEQ ID NO:43), GGMGM (SEQ IDNO:44), GGMMG (SEQ ID NO:45), MGGMG (SEQ ID NO:46), MGMGG (SEQ IDNO:47), GMMGM (SEQ ID NO:48), MGMMG (SEQ ID NO:49), GMGGM (SEQ IDNO:50), MMGMG (SEQ ID NO:51), GMMGG (SEQ ID NO:52), GMGGG (SEQ IDNO:53), GGMGG (SEQ ID NO:54), GGGMG (SEQ ID NO:55) and GGGGG (SEQ IDNO:56).
 4. The pharmaceutical formulation of claim 3, wherein theoligopeptide is bound to the N-terminal of IL-7.
 5. The pharmaceuticalformulation of claim 2, wherein the IL-7 is a polypeptide having theamino acid sequence of any one of SEQ ID NOs: 1 to
 6. 6. Thepharmaceutical formulation of claim 2, wherein the Fc region of theimmunoglobulin is bound to the C-terminal of IL-7.
 7. The pharmaceuticalformulation of claim 6, wherein the Fc region of the immunoglobulin hasthe amino acid sequence of SEQ ID NO:7.
 8. The pharmaceuticalformulation of claim 1, wherein the basal buffer is histidine-acetate orsodium citrate.
 9. The pharmaceutical formulation of claim 1, whereinthe sugar is selected from the group consisting of sucrose, trehalose,dextrose, and a mixture thereof.
 10. The pharmaceutical formulation ofclaim 1, wherein the surfactant is selected from the group consisting ofpolysorbate, polyoxyethylene alkyl ether, polyoxyethylene stearate,alkyl sulfates, polyvinyl pyridone, poloxamer and a mixture thereof. 11.The pharmaceutical formulation of claim 1, further comprising any oneamino acid selected from the group consisting of arginine, glutamate,glycine, histidine, and a mixture thereof.
 12. The pharmaceuticalformulation of claim 11, wherein the concentration of the amino acidranges from 40 to 60 mM.
 13. The pharmaceutical formulation of claim 12,wherein the concentration of the amino acid is 50 mM.
 14. Thepharmaceutical formulation of claim 1, further comprising a sugaralcohol of 1 to 2 w/v %.
 15. The pharmaceutical formulation of claim 14,wherein the sugar alcohol is selected from the group consisting ofsorbitol, xylitol, maltitol, mannitol, and a mixture thereof.
 16. Thepharmaceutical formulation of claim 1, wherein the pH of the formulationis 5.0.
 17. The pharmaceutical formulation of claim 1, wherein theformulation is a liquid formulation.